1. Technical Field
The present invention relates to a synthetic, plasma-free suspension medium for red blood cells and platelets. More particularly, the present invention relates to a synthetic preservation medium for red blood cells or platelets which (1) is free of blood plasma and other proteins, (2) extends red blood cell or platelet shelf life while maintaining the quality of concentrates of these components during storage for transfusion and (3) is free of unessential organic compounds.
2. State of the Art
Blood is composed of two major portions. These portions can be recognized when a specimen of blood is drawn and clotting is prevented. That portion of the blood which settles to the bottom of the vessel holding the specimen is termed the "formed elements." The formed elements comprise red blood cells and other particulate components such as white blood cells, red blood cells, and platelets. Platelets are also known as thrombocytes. The formed elements are characteristically 40 to 50 percent of the bulk of normal human blood. The cloudy liquid which does not settle in a blood specimen is the portion of the blood known as plasma. Plasma is primarily water, but contains inorganic and organic substances as well as dissolved gases and miscellaneous foreign substances. The inorganic substances contained in blood plasma are primarily electrolytes. The most significant of these electrolytes are presented in Table 1 in the concentrations typically found in healthy, human blood plasma.
TABLE 1 ______________________________________ Sodium 142.0 mEq/l Potassium 4.3 mEq/l Calcium 5.0 mEq/l Magnesium 3.4 mEq/l Chloride 104.0 mEq/l Bicarbonate 27.0 mEq/l Phosphate 2.3 mEq/l Sulfate 0.6 mEq/l ______________________________________
The most significant organic substances found in the plasma are lactic acid, urea, amino acids, creatinine, glucose, hormones, proteins, albumins, and globulins.
Modern medicine has been developing solutions that are added to blood in vivo and/or mixed with blood in vitro. Products that are used for adding to blood in vivo are primarily used for intraveneous feeding, pharmaceutical vehicles, and/or electrolyte replacement in patients who are bedfast. These solutions are primarily comprised of water that contains dextrose and, optionally, electrolytes. Dextrose is typically present in these solutions in about a 5 percent concentration and provides a nutrient for blood cells or tissue cells. The electrolytes contained in these solutions vary widely. The solutions that contain electrolytes that most closely resemble blood plasma contain a plurality of the electrolytes presented in Table 1. A specific example of a dextrose and electrolyte solution suitable for in vivo addition in blood is Locke-Ringer's solution. The formula for Locke-Ringer's solution is presented in Table 2.
TABLE 2 ______________________________________ Reagent Sodium Chloride 9.0 Gm Reagent Potassium Chloride 0.42 Gm Reagent Calcium Chloride 0.24 Gm Reagent Magnesium Chloride 0.2 Gm Sodium Bicarbonate 0.5 Gm Dextrose 0.5 Gm Water, recently distilled from a 1000 ml hard glass flask, in a sufficient quantity, to make ______________________________________
Other solutions suitable for the addition of blood in vivo can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, 14th Edition (1970), pages 815 to 847.
Solutions that are added to blood in vitro are either simple anticoagulant nutrient mixtures principally designed for preserving whole blood or separated components for limited periods of time or are specific nutrient solutions optimized for prolonged preservation of specific blood components such as red cells, platelets, white blood cells, or mixtures of these components. When platelets are included as one component of blood that is to be collected and preserved in vitro with red blood cells, it is desirable to use a less active anticoagulant.
The most frequently used anticoagulant added to collected whole blood is known as "acid citrate-dextrose" or "ACD". This anticoagulant solution contains a mixture of anticoagulant and nutrient specifically; (1) citric acid and sodium citrate in optimum concentrations sufficient to chelate calcium without creating an unphysiological pH; and (2) dextrose in concentrations sufficient for short term preservation of blood or components, especially the red blood cells. A less acidic solution that has been found desirable to preserve both whole blood and other cellular fractions is known as "anticoagulant citrate-phosphate-dextrose solution" or "CPD". The components of anticoagulant citrate phosphate-dextrose solution are presented in Table 3.
TABLE 3 ______________________________________ Citric Acid (anhydrous) 3.0 Gm Sodium Citrate (dihydrate) 26.3 Gm Sodium Biphosphate (monohydrate; 2.22 Gm NaH.sub.2 PO.sub.4 H.sub.2 O) Dextrose 25.5 Gm Water for Injection, in a 1000 ml sufficient quantity to make ______________________________________
Specific elements of the particulate component of blood can be separated and better preserved for later transfusion at different temperatures. Simple separation processes can be used to collect and preserve white blood cells and platelets which are best stored at 22.degree. C. rather than 4.degree. C. which is optional for red cells.
Storage of red cells at 4.degree. C. for transfusion results in deterioration of the cells over a period of time. This deterioration of the cells is termed "storage lesion" and is characterized by increased hemolysis and decreased viability resulting in lowered post-transfusion survival of the red blood cells. The alterations associated with storage lesion include intracellular changes such as decreased potassium ion and increased sodium and calcium ion concentrations, the loss of 2,3-diphosphoglycerate (2,3 DPG) resulting in altered oxygen-transfer characteristics, the depletion of adenosine triphosphate (ATP), and membrane skeleton alterations resulting in decreased deformability and increased fragility.
Red cells were previously, typically stored in a combined nutrient-anticoagulant mixture consisting of 1 part nutrient-anticoagulant to 7 parts plasma (acid-citrate-dextrose). The progression of storage lesion was the limiting factor to the shelf life of stored red cells. The shelf life of stored red blood cells was generally limited to 21 days.
More recently, improved red cell viability with storage periods up to 49 days has been achieved with additives specifically mixed with red cells after the plasma and platelets have been removed. Typically, these additive solutions are a simple saline solution fortified with adenine, glucose, and other, minor constituents that are added to retard development of hemolysis and loss of 2,3 DPG. The primary container used with these additive solutions contains only a simple anticoagulant which for convenience has been CPO as described previously. After collection, the anticoagulated whole blood is centrifuged and the platelet-rich plasma and red blood cells are transferred to a satellite bag. The red cell additive solution is then mixed with the red cells. Mixing the additive solution into the separated red blood cells prevents contamination of other separated components of the blood with the red cell additive solution. Desirable flow properties can be maintained for the red cells by using an appropriate volume of the additive solution.
Numerous additive solutions for red blood cells exist either as commercial or research products. The typical additive solution for red blood cells is derived from a saline-adenine-glucose (SAG) medium. SAG was developed in Sweden in the mid 1970's and provides acceptable erythrocyte survival after 35 days of storage rather than the 21 days that was previously available with combined anti-coagulant nutrient solutions SAG maintains acceptable ATP levels throughout 35 days of storage. The storage of red blood cells for 35 days in SAG results in an average hemolysis of approximately 1 percent by the last day of storage which was much greater than the 0.1 percent normally associated with either CPD or ACD. The increased hemolysis of red blood cells in SAG, when compared to storage in a simple solution of sodium citrate, dextrose, citric acid, monobasic sodium phosphate, which is now fortified with adenine (CPDA-1) for prolonged storage (35 days), is believed to result from the action of either white cell proteases upon the erythrocyte membrane in the absence of plasma proteins or as a result osmotic destabilization due to unphysiological storage solutions.
A modified SAG contains mannitol (29 mM) and is known by the symbol SAG-M. This additive solution results in membrane stabilization and decreased hemolysis. A product having a similar formula to SAG-M is the ADSOL.RTM. brand of storage medium. ADSOL.RTM. which is licensed for commercial use in the United States and contains 50 percent more adenine and 150 percent more glucose in addition to 750 mg/dl of mannitol than does SAG-M. This product is produced by Baxter, Inc., 1425 Lake Cook Road, Deerfield, Ill. 60015. The ingredients of the ADSOL.RTM. brand of storage medium are presented in Table 4.
TABLE 4 ______________________________________ Dextrose 2.2 g Sodium Chloride 900.0 mg Mannitol 750.0 mg Adenine 27.0 mg Water in a sufficient 100.0 ml quantity to make ______________________________________
The presence of mannitol in the ADSOL.RTM. brand storage medium reduces hemolysis even in the presence of leukocyte proteases. The high concentration of dextrose in the ADSOL.RTM. brand storage medium is believed to have desirable effects on other red blood cell parameters. The ADSOL.RTM. brand storage medium can effectively store red cells for 42 days.
However, there are disadvantages and problems with the current generation of red blood cell storage media. These media are ionically unbalanced with sodium as the only cation. Since the active cation transport system of red blood cells is inhibited at low temperatures, a loss of the normal concentration gradient occurs in the red blood cells due to passive diffusion of the cations. This can cause swelling of the cell and eventual lysis. The addition to these solutions of a membrane-stabilizer or "osmotic agent" such as mannitol or sorbitol can not fully compensate for the environmental perturbation of the red cells in these solutions.
The present generation of red blood cell additive solutions have a limited buffering capacity. These limited buffering systems allow the pH of the red blood cell concentrates to decline to levels below 6.5 after about 42 days of storage at 4.degree. C. At this pH level and below, glycoysis with ATP reduction causes the loss of red blood cell viability during storage. The 2,3 DPG level is also markedly sensitive to a decline in pH during storage. Loss of 2,3 DPG results in altered red blood cell oxygen binding characteristics.
The storage of platelets in special additive solutions is commonly performed in special or "second generation" containers. These containers are more gas permeable than the "first generation" of blood storage containers that are made of polyvinyl chloride (PVC). The use of these special containers allows carbon dioxide (CO.sub.2) gas to escape during storage of the blood component. The escape of CO.sub.2 gas reduces the formation of carbonic acid in the preservation of storage solution. The reduction of carbonic acid in solution extends the period of time during which a physiologically acceptable pH can be maintained in the storage solution.
U.S. Pat. No. 4,447,415 to Rock et al. discloses a liquid storage medium for platelets that is plasma-free. The medium of this invention uses one or more additives in conjunction with a saline and anticoagulant, dextrose-containing solution that is desirably a form of CPD Tyrode's solution. The additives disclosed as being suitable for use with this invention include (1) reversible inhibitors that are organic compounds such as indomethacin, quinacrine, or vitamin E and (2) substances to raise cyclic adenosine monophosphate levels such as prostaglandins E.sub.1, D.sub.2, or I.sub.2. Many of these additives fail to meet safety and regulatory requirements required for substances for infusion into humans and are, therefore, only suitable for experimental use or only for in vitro use. Other additives disclosed as suitable for use with this invention include (1) nutrients such as fructose and other sugars, adenine, or acetyl CoA and (2) buffers such as phosphate and certain amino acids. The organic compounds or additives identified as nutrients do not eliminate the requirement for the presence of dextrose in the medium. The combination of additive and glucose carried over in the plasma satisfies the nutrient requirement for the platelets for periods of storage time extending up to 5 days and the additives identified as buffers cannot maintain a balanced pH during extended platelet storage periods beyond this time. Since the glucose levels are clearly-inadequate for red cell storage over longer time periods and these buffers cannot adequately buffer the amount of lactic acid produced by viable, suspended red cells stored at temperatures of 4.degree..+-.2.degree. C. cell death due to pH fall is likely. As a result this solution, though potentially acceptable for platelets, cannot be used to store red cells. This disclosure is, therefore, not directed to a storage medium for red blood cells.
The industry is lacking a blood cell storage additive medium that is free of plasma and organic compounds, which can be used to store red blood cells and platelets for extended periods of time and which is safe for in vivo human use.